RNA serves as an essential component of every modern biological study. It provides a raw material for medical diagnostics, drug design, recombinant protein production, bioinformatics and almost every area concerning the pharmaceutical and biotechnology industries.
RNA is an essential and universal component of all organisms. There are three major types of RNA; these are messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA), the latter being the most common type. In addition, some viruses encode their genes in the form of RNA such as the retroviruses, HIV being one example of this type. Other RNA forms include small infective RNA loops called viroids, PSTV being one example of this type. RNA has many diverse functions such as in the production of proteins and the storage of genetic information. The ability of RNA to carry out these functions is dependent on its composition and sequence.
mRNA is naturally produced from a DNA template by a process known as transcription. It accounts for less than 5% of the total RNA in a cell and exists in hundreds of thousands of forms depending on its sequence; however, nearly all eukaryotic mRNA have a 5′ CAP structure and a 3′ poly (A)+ tail, the latter serving as an essential feature for purifying mRNA from the bulk of the cellular RNA. There are estimated to be 500,000 mRNA molecules in an average mammalian cell. It contains the coding region for a protein and is crucial to understand the function of a gene.
All RNA molecules are linear macromolecules composed of repeated monomers (ribonucleotides) comprising a base, a ribose sugar and a phosphate. There are four principal bases: uracil, cytosine, guanine and adenine; the order in which they are connected together, the sequence, leads to many of the unique properties of RNA.
RNA differs chemically from DNA in two major ways. Firstly, it contains uracil instead of thymine, and secondly, RNA has a 2′-OH group on the ribose sugar instead of 2′-H found on the deoxyribose sugar of DNA.
Natural RNA has the 2′ carbon atom bonded to two other carbon atoms (C1′ and C3′), a hydrogen atom and an oxygen atom that forms part of a hydroxyl group (here called the 2′-OH group). The 2′-OH group endows RNA with many of its unique properties such as structure, reactivity and instability. The 2′-OH group can also assist in the cleavage of the phosphodiester bonds between ribonucleotides leading to chain cleavage and hence RNA degradation.
When RNA is manipulated for any number of common laboratory practices, its inherent instability leads to considerable technical and experimental difficulties. For example, measuring the abundance and size of a particular mRNA species is frequently considered essential to understanding the function of a gene. When the particular mRNA under study is degraded, even to a small extent, such measurements become impossible to carry out reliably or accurately. Another example would be the synthesis of a cDNA copy of a mRNA, where degradation of the mRNA precludes any possibility of obtaining a full and representative cDNA. Such cDNA copies are considered essential experimental tools because they allow a full and accurate characterisation of the gene such as its pattern of expression and chromosomal location. Furthermore the cDNA is essential to produce recombinant protein.
Protecting RNA from degradation whilst maintaining its biological activity is an essential task for any researcher or technician. However, the difficulty of removing nuclease activity from the RNA and the ease of accidentally introducing it, often precludes successful RNA manipulation to all except the most experienced. The cost and time considerations of RNA shipping and storage, equipment sterilisation, purchase of disposable plastic ware, training personnel and repeating failed experiments are a significant part of any laboratory budget.
The most important aspect of purifying RNA is to prevent degradation by RNases. RNases can be introduced from three sources: (1) intra-cellular sources due to carry-over from the experimental sample, (2) from external sources such as the researcher's skin secretions and (3) purified RNase used for DNA purification. RNases are truly ubiquitous; they can be found in finger tip secretions, dust, microbes, nearly all biological materials and even slight contamination will inevitably lead to RNA degradation. Compounding the problem is the common use of highly concentrated RNase in many DNA purification kits.
There are two principal means by which the 2′-OH group of ribonucleotides can be modified (a) enzymatically and (b) chemically. Enzymatic modification of the 2′-OH group arises from highly specific enzyme-catalysed reactions. For example, ribonucleotide reductase modifies the monomer ribonucleoside diphosphate, whereas an entire RNA molecule will not be recognised as a substrate. Another example is the methyl transferases that use an entire RNA molecule as a substrate but modify only a few 2′-OH groups per molecule.
The chemical synthesis of RNA and DNA is well known and many companies provide custom RNA and DNA synthesis (for review, see Eaton, (1995) Annu. Rev. Biochem. 64, 837). A considerable body of published work exists describing the different approaches to its synthesis (for review, see: Usman and Cedergreen (1992) TIBS 17:334). Protective groups have been reviewed (Greene and Wuts (1991) Protective Groups in Organic Synthesis, 2nd Ed. Wiley Interscience). The most prominent route for preparation of 2′-modified ribopyrimidines is through the introduction of nucleophiles to the corresponding 2,2′-anhydropyrimidine precursor. This reaction is limited to preparation of 2′-halides, 2′-azide, 2′-thiolates (Moffatt, (1979) In: Nucleoside Analogues, Ed. Walker, pp.71-163, NY, Plenum., Townsend, (1988) Chemistry of Nucleosides and Nucleotides, pp.59-67, NY, Plenum), 2′-azido (Verheyden, et al., (1971) J. Org. Chem. 36:250) and 2′-amino ribonucleoside (Wagner, et al., (1972) J. Org. Chem. 37:1876). Methylation of the 3′, 5′-protected precursor gives 2′-O-methyl ribonucleosides (Sproat, et al., (1991) Oligonucleotides and Analogues: A Practical Approach, ed. F. Eckstein, pp.49-86, NY. Oxford Univ. Press), and similarly 2′-O-alkyl and 2′-O-allyl derivatives have been made (Sproat, (1991) Nucleic Acids Res. 19:733, Lesnik, et al., (1993) Biochemistry. 32, 7832). Other modifications include 2′-methyl (Matsuda, et al., (1991) J. Med. Chem. 34:234), 2′-phenyl, 2′-alkyl ribonucleosides (Schmit (1994) Synlett. 234), 2′-acetylated (Imazawa, et al., (1979) J. Org. Chem. 44:2039), 2′-fluoro, 2′-trifluoromethyl (Schmit, (1994) Synlett. 241), 2′-mercapto (Imazawa, et al., (1975) Chem. Pharm. Bull. 23:604) and 2′-thio ribonucleosides (Divakar, et al., (1990) J. Chem. Soc. Perkin Trans. 1:969). 2′-Fluoro, 2′-O-methyl, 2′-O-propyl and 2′-O-pentyl nucleotides have each been incorporated into oligoribonucleotides (Cummins, (1995) Nucleic Acid Res. 23:2019). In each case the substrates and products are non-polymerised, that is they exist as simple monomers and not in the polyribonucleotide (RNA) form.
Practical applications of such 2′-modified ribonucleotides and polyribonucleotides include anti-viral activity (Wohlrab, et al., (1985) Biochem. Biophys. Acta 824:233), inhibition of bacterial growth (Salowe, et al., (1987) Biochem. 26:3408) and antisense oligonucleotides (Pieken, et al., (1991) Science 253:314). It has been shown that 2′-O-methoxyethyl replacement of the 2′-OH group can provide favourable conformations to enhance its binding to a target RNA. Research applications include developing novel ligands by the SELEX (systematic evolution of ligands by exponential enrichment) procedure (Gold, et al., (1995) Annu. Rev. Biochem. 64:763) and ribozyme research (Uhlenbeck, et al., (1987) Nature 328:596). The modification of the 2′-OH group as an investigative tool has been reviewed (Heidenreich, (1993) FASEB J., 7:90). Many of the 2′-modified ribonucleotide triphosphates (Amersham International, Buckinghamshire, UK) or polymers (Midland Certified Reagent Company, Texas, USA) are available commercially.
Procedures suitable for modifying the 5′-OH and 3′-OH groups of deoxyribose have been developed in order to facilitate DNA oligonucleotide synthesis. For example, acetic anhydride in the presence of N-methylimidazole and tetrahydrofuran composes what is called the ‘capping’ reagent used commonly in almost all automated DNA synthesisers today. Other applications for acetic anhydride have been found, for example in the production of L-nucleoside dimers (Weis, International Patent Application, WO 97/11087).
Chemical modification studies are routinely carried out in order to analyse protein-RNA interactions (Jones et al., (1994) in RNA Isolation and Analysis. Bios. Oxford; Hecht (1996) Bioorganic Chemistry Nucleic Acids, Oxford University Press). Chemical modification is usually carried out with diethyl pyrocarbonate (Green et al., (1995) J. Mol. Biol. 247:60) which modifies the purine base or hydrazine which cleaves pyrimidines. For DNA footprinting studies, ethylnitrourea treatment is used to modify the phosphates leading to ethyl phosphotriester formation (Siebenlist and Gilbert (1980) Proc. Nat'l. Acad. Sci. 77:122; Green et al., (1995) J. Mol. Biol. 247:60). Alternatively, DNA may be treated with dimethylsulfate which leads to alkylation on the base (Carey (1989) J. Biol. Chem. 264:1941).
Modification of RNA chains using chemical reagents has been reported in several articles. Specific modifying chemicals that have been used include dimethylsulphate leading to base modification (Bollack et al., (1965) Bull. Soc. Chim. Biol. 47:765-784), N-chlorosuccinimide leading to base modification and RNA degradation (Duval and Ebel, (1967) Bull. Soc. Chim. Biol. 49:1665-1678; Duval and Ebel., (1966) C.R. Acad. Sc. Paris t. 263:1773 series D), N-bromosuccinimide (Duval and Ebel, (1965) Bull. Soc. Chim. Biol. 47:787-806), diazomethane leading to methylation of the base and phosphate causing RNA breakdown (Kriek and Emmelot., (1963) Biochemistry 2:733), carbodiimide leading to base modification (Augusti-Tocco and Brown (1965) Nature 206:683), alkyl halides leading to base and phosphate modification (Ogilvie et al., (1979) Nucleic Acids Res. 6:1695) and allyl bromide leading to guanine modification and chain degradation (Bollack and Ebel, (1968) Bull. Soc. Chim. Biol. 50:2351-2362). It has been reported that the use of acetic anhydride in DMF results in acylation of cytosine (Keith and Ebel (1968) C.R. Acad. Sc. Paris t. 266:1066 series D). Methyl sulphate has been used to modify the bases of an RNA template (Louisot et al., (1968) Annales de L'institut Pasteur. 98). The results of such chemical modification reactions of RNA are therefore degradation, base and/or phosphate modification.
Other work has shown the acylation of the base uridine of tRNA (Glu) using benzoic anhydride but not the 2′-OH groups (Cedergreen et al., (1973) Biochem. 12:4566-4570). Using benzoic anhydride, phthalic anhydride, N-benzoylimidazole and acetylimidazole Cedergreen demonstrated that there is only one major site of modification of the tRNA and that 1 mole of anhydride reacted with 1 mole of tRNA. The authors conclude that acylation occurred on the base moiety.
The free amino function of the base is often N-acylated when the nucleotide or nucleoside is treated with an anhydride such as acetic anhydride or an acid chloride in anhydrous pyridine. Indeed this method is often used to protect the amino groups of nucleosides (Brimacombe et al., (1968) Czech. Chem. Commun. 33:2074; Saneyoshi (1968) Chem. Pharm. Bull. 16:1400; Cedergreen et al., (1971) 49:730; Amarnath and Broom., (1977) Chem. Rev. 77:183). Furthermore it has been shown that RNA treated with acetic anhydride in dimethyl formamide is specifically modified on the cytosine base and not on the 2′-OH group (Keith and Ebel, (1968) Biochim. Biophys. Acta. 166:16-28). It is well known and widely reported that ribonucleotides in pyridine solution exclusively acetylate the base.
The work of Chang and Lee (Biochemistry (1981) 20:2657) demonstrated the methylation of RNA using methyl methanesulfonate. Six methylation sites were identified, 5 on the bases and one on the phosphate.
This body of work, taken together, strongly suggests that chemical treatment of nucleic acids would be likely to result in the modification of either the bases or the phosphate with or without RNA degradation. This is not surprising considering the chemical reactivity of these groups. Obtaining 2′-OH regiospecific modification of RNA is the basis for this invention.
(2′-azido-2′-deoxyuridylic acid) has been prepared (Torrence, (1972) J. Amer. Chem. Soc. 94:3638-3639). Pyridine-catalysed quantitative examples of acetylation are reported for 3′-hydroxynucleotides (Weber and Khorana, (1972) J. Mol. Biol. 72:219; Zhdanov and Zhenodarova, (1975) Synthesis 222).
The acetylation procedure was first described by Khorana and co-workers (Stuart and Khorana (1963) J. Biol. Chem. 85:2346) who acetylated the terminal 3′-OH group of deoxyribonucleotides and oligonucleotides with acetic anhydride. No modification of the bases was observed unless the acetylation was carried out in the presence of strongly basic solvents such as pyridine or tributylamine (Michelson and Grunberg-Manago, (1964) Biochem. Biophys. Acta, 91:92).
Acetylation of a tRNA molecule was carried out by using acetic anhydride. A change in the secondary structure was reported (Knorre, et al., (1965) Biokhimiya 30:1218). Modification of 30% of the 2′-OH groups of tRNA was found to destroy its secondary structure. Further work by the same researchers demonstrated that variable acetylation levels of tRNA (Knorre, et al., (1966) Biokhimya 31:1181) and polyribo-oligonucleotides (Knorre, et al., (1967) Biochim. Biophys. Acta 142:555) could be achieved by use of acetic anhydride and N,N-dimethylformamide. It was also shown that acetylated poly(U) lost its ability to hydrogen bond with poly(A). Acetylated forms of poly(U) and poly(A) were reportedly quite unable to direct polypeptide synthesis in a cell-free system (Knorre, et al., (1967) Molekul. Biol. 1:837).
More recently, it has been reported in a publication that mRNA from a cell-free transcription system has been used as a substrate for acetylation (Ovodov and Alakhov, (1990) FEBS 270:111). Acetylation of 70-75% of the 2′-OH groups was said to be achieved using the method of Knorre et al. However, results presented in the publication suggest otherwise. FIG. 9 shows no change in mobility indicating that no modification actually took place.